Alteration of proteolytic cleavage of botulinum neurotoxins

Abstract

The present invention pertains to a polynucleotide encoding a modified neurotoxin polypeptide comprising a modified neurotoxin light chain and a heavy chain, said modified light chain having at least one modification conferring altered cleavage by calpain proteases. Further encompassed by the present invention are vectors and host cells comprising the polynucleotide of the invention as well as polypeptides encoded by the said polynucleotide. In addition, the invention relates to compositions comprising the polynucleotide, vector, host cell or polypeptide of the invention as a medicament.

1 - 16 . (canceled) 
     
     
         17 . A polynucleotide encoding a modified neurotoxin polypeptide comprising a modified neurotoxin light chain and a heavy chain, wherein the modified neurotoxin light chain exhibits at least one modification conferring altered cleavage by calpain proteases. 
     
     
         18 . The polynucleotide of  claim 17 , wherein the at least one modification confers an increased cleavage by calpain proteases compared to a non-modified neurotoxin light chain. 
     
     
         19 . The polynucleotide of  claim 18 , wherein the at least one modification is at least one calpain cleavage site which has been introduced into the neurotoxin light chain. 
     
     
         20 . The polynucleotide of  claim 19 , wherein the modified neurotoxin light chain and the heavy chain are derived from BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/F, or BoNT/G. 
     
     
         21 . The polynucleotide of  claim 17 , wherein the modified neurotoxin polypeptide exhibits a shortened duration of biological activity. 
     
     
         22 . The polynucleotide of  claim 17 , wherein the at least one modification confers a decreased cleavage by calpain proteases compared to a non-modified neurotoxin light chain. 
     
     
         23 . The polynucleotide of  claim 22 , wherein the at least one modification is at least one substitution within a calpain cleavage site in the neurotoxin light chain. 
     
     
         24 . The polynucleotide of  claim 23 , wherein the at least one substitution is a substitution at the P1, P2, P3, P4, P5, P1′, P2′, and/or P3′ position of the calpain cleavage site. 
     
     
         25 . The polynucleotide of  claim 23 , wherein the modified neurotoxin light chain and the heavy chain are derived from BoNT/A, BoNT/B or BoNT/C1. 
     
     
         26 . The polynucleotide of  claim 22 , wherein the modified neurotoxin polypeptide exhibits a prolonged duration of biological activity. 
     
     
         27 . A vector comprising the polynucleotide of  claim 17 . 
     
     
         28 . A host cell comprising the polynucleotide of  claim 17 . 
     
     
         29 . A host cell comprising the vector of  claim 27 . 
     
     
         30 . A polypeptide encoded by the polynucleotide of  claim 17 . 
     
     
         31 . A pharmaceutical composition comprising the polypeptide of  claim 30 . 
     
     
         32 . A method for treating and/or preventing a disease or disorder comprising administering to a subject in need thereof a therapeutically effective dose of a composition comprising the polynucleotide of  claim 17  or a polypeptide encoded thereby. 
     
     
         33 . The method of  claim 32 , wherein the disease or disorder is selected from the group consisting of: wound healing, immobilisation for bone and tendon fracture treatment, post surgery immobilization, post surgery immobilization after haemorrhoidectomy, post surgery immobilization after introduction of dental implants, post surgery immobilization after hip joint replacement (endoprothesis), post surgery immobilization after knee arthroplasty, post surgery immobilization after ophthalmological surgery, acne, irritable bowel disease and prostate hyperplasia. 
     
     
         34 . A method for treating and/or preventing a disease or disorder comprising administering to a subject in need thereof a therapeutically effective dose of a composition comprising the polynucleotide of  claim 22  or a polypeptide encoded thereby. 
     
     
         35 . The method of  claim 34 , wherein the disease or disorder is selected from the group consisting of: voluntary muscle strength, focal dystonia, cervical dystonia, cranial dystonia, benign essential blepharospasm, hemifacial spasm, focal spasticity, gastrointestinal disorders, hyperhidrosis, cosmetic wrinkle correction, blepharospasm, oromandibular dystonia jaw opening type, oromandibular dystonia jaw closing type, bruxism, Meige syndrome, lingual dystonia, apraxia of eyelid, opening cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, spasmodic dysphonia/adductor type, spasmodic dysphonia/abductor type, spasmodic dyspnea, limb dystonia, arm dystonia, task specific dystonia, writer's cramp, musician's cramps, golfer's cramp, leg dystonia, thigh adduction, thigh abduction knee flexion, knee extension, ankle flexion, ankle extension, equinovarus, deformity foot dystonia, striatal toe, toe flexion, toe extension, axial dystonia, pisa syndrome, belly dancer dystonia, segmental dystonia, hemidystonia, generalised dystonia. dystonia in corticobasal degeneration, dystonia in lubag, tardive dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hallervorden-Spatz disease, dopa-induced dyskinesias/dopa-induced dystonia, tardive dyskinesias/tardive dystonia, paroxysmal dyskinesias/dystonias, kinesiogenic non-kinesiogenic action-induced palatal myoclonus, myoclonus myokymia, rigidity, benign muscle cramps, hereditary chin trembling, paradoxic jaw muscle activity, hemimasticatory spasms, hypertrophic branchial myopathy, maseteric hypertrophy, tibialis anterior hypertrophy, nystagmus, oscillopsia supranuclear gaze palsy, epilepsia, partialis continua, planning of spasmodic torticollis operation, abductor vocal cord paralysis, recalcitrant mutational dysphonia, upper oesophageal sphincter dysfunction, vocal fold granuloma, stuttering, Gilles de la Tourette syndrome, middle ear myoclonus, protective larynx closure, postlaryngectomy, speech failure, protective ptosis, entropion sphincter Odii dysfunction, pseudoachalasia, nonachalsia, oesophageal motor disorders, vaginismus, postoperative immobilisation tremor, bladder dysfunction, detrusor sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, crow's feet, frowning facial asymmetries, mentalis dimples, stiff person syndrome, tetanus prostate hyperplasia, adipositas, treatment infantile cerebral palsy strabismus, mixed paralytic concomitant, after retinal detachment surgery, after cataract surgery, aphakia myositic strabismus, myopathic strabismus. dissociated vertical deviation, strabismus surgery, esotropia, exotropia, achalasia, anal fissures, exocrine gland hyperactivity, Frey syndrome, Crocodile Tears syndrome, hyperhidrosis, axillar palmar plantar rhinorrhea, relative hypersalivation in stroke, Parkinson's Disease, amyotrophic lateral sclerosis, spastic conditions, encephalitis and myelitis autoimmune processes, multiple sclerosis, transverse myelitis, Devic syndrome, viral infections, bacterial infections, parasitic infections, fungal infections, hereditary spastic paraparesis, postapoplectic syndrome hemispheric infarction, brainstem infarction, myelon infarction, central nervous system trauma, hemispheric lesions, brainstem lesions, myelon lesion, central nervous system haemorrhage, intracerebral hemorrhage, subarachnoidal hemorrhage, subdural hemorrhage, intraspinal hemorrhage, neoplasias, hemispheric tumors, brainstem tumors, myelon tumor and vaginism.